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Improved Compensation in Flow Cytometry

Conventional compensation of flow cytometry (FMC) data of an N-stained sample requires additional data sets, of N single-stained control samples, to estimate the spillover coefficients. Single-stained controls however are the least rigorous controls because any of the multi-stained controls are closer to the N-stained sample. We have developed a new, optimization-based, compensation method that is able to use not only single- but also multi-stained controls to improve estimates of the spillover coefficients. The method has been demonstrated on a data set from 5-stained dendritic cells (DCs) with 5 single-stained and 8 multi-stained controls. This approach is practical and leads to significant improvements in FCM compensation.